Anti-proliferation domain of a human Bcl-2 and DNA encoding the same

ABSTRACT

A domain of Bcl-2 that suppresses apoptosis by allowing cell survival permits cell proliferation when mutated. The wild type domain includes amino acid residues 51 to 97 (SEQ ID NO:13) of Bcl-2. Peptides including the domain and nucleotides encoding the domain are useful in molecular screening of human tumors for the presence of mutations that allow proliferation of cells that were otherwise marked for apoptosis. The peptides are also useful to screen for proteins that play a role in the modulation of cellular proliferation.

This invention was made with government support under grants CA-33616and CA-31719 from the National Cancer Institute. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of cell physiology,and more particularly to tumorigenesis and to apoptosis, i.e. programmedcell death. The novel peptides and nucleotides of the invention areuseful in molecular screening of human tumors for the presence ofmutations that allow the proliferation of cells that were otherwisemarked for apoptosis. The novel peptides and nucleotides are also usefulto screen for proteins that play a role in the modulation of cellularproliferation.

BACKGROUND OF THE INVENTION

The bcl-2 gene was discovered as typically involved in the t(14;18)chromosomal translocations observed in human follicular lymphoma (1-3).This chromosomal rearrangement results in deregulated high-levelexpression of the bcl-2 gene. In addition, Bcl-2 is also expressed atelevated levels in a variety of other tumors (4-6). The Bcl-2 proteinsuppresses apoptosis induced by a multitude of stimuli (7,8).Suppression of apoptosis by Bcl-2, while allowing cell survival, ischaracterized by growth arrest associated with Bcl-2 activity (40).Although bcl-2 was discovered as a candidate oncogene, conventionaltransformation assays indicate that it does not possess dominantoncogenic activity (9). It is therefore believed that unlike otheroncogenes, bcl-2 contributes to oncogenesis primarily by extending cellviability, thereby perturbing the homeostatic mechanisms that controlcell number and by providing an environment for other genetic changes(10).

In spite of a lack of detectable autonomous transforming activity, bcl-2has been shown to synergize with c-myc in the generation of malignantcells (11). Since constitutive expression of c-myc induces apoptosisunder certain conditions (12-14) that can be suppressed by Bcl-2(14-16), it appears that the c-myc-cooperating oncogenic activity ofbcl-2 may be related to its anti-apoptosis activity. In addition, Bcl-2can also efficiently suppress apoptosis induced by tumor suppressorproteins such as p53 (17-21). This suggests that Bcl-2 may contribute tooncogenesis by suppressing apoptosis induced by oncogenes and tumorsuppressor genes.

Although mutations within the Bcl-2 protein that permit proliferation ofcells that would otherwise undergo total apoptosis could play a moredirect role (as opposed to deregulated expression) in oncogenesis, thusfar no such mutants have been identified in naturally arising tumors orunder experimental conditions.

SUMMARY OF THE INVENTION

The present inventor here describes the identification andcharacterization of a hitherto unrecognized domain within human Bcl-2,which the inventor has designated the "anti-proliferation (AP) domain",that is required for the proliferation-restraining activity of Bcl-2.Mutants in this domain of Bcl-2 are described that retain the ability tosuppress apoptosis induced by the p53 tumor suppressor protein and Myconco-protein, while allowing concomitant cell proliferation.

More specifically, the present inventor has identified a deletion mutantof Bcl-2 that has a novel activity. The deletion mutant, designatedBcl2Δ51-85, not only suppresses apoptosis induced by the tumorsuppressor protein p53 and the Myc onco-protein, but unlike wt Bcl-2,permits continued cell proliferation. These results may have importantimplications for oncogenesis involving Bcl-2. Unlike other oncogenes,the bcl-2 proto-oncogene promotes cell survival without significant cellproliferation. These results suggest that certain mutations caninactivate a proliferation-restraining activity. Further, the observedeffect against oncogene/anti-oncogene-induced apoptosis may potentiallyprove to be of considerable significance in oncogenic events involvingBcl-2. Such inactivating mutations within the non-conserved region ofBcl-2 may enhance tumorigenesis by antagonizing the apoptotic activitiesof p53 and Myc as well as by permitting continued cell proliferation.

The molecular basis for the loss of proliferation-restraining activityin the Bcl-2 mutant has been partially elucidated as described inExample 3. The results suggest that the loss of activity does notcorrelate with the ability of Bcl-2 to interact with several proteins.However, the interaction between the Bcl-2 mutant and thedeath-promoting protein Bax appears to be enhanced compared to theinteraction of Bax with wild type Bcl-2. It is not clear whether thisenhancement is due to an increased affinity of the Bcl-2 mutant for Baxor increased stability of the Bcl-2/Bax complex. The importance of theBcl-2/Bax interaction to the proliferation-restraining function of Bcl-2is unknown.

Also, the region deleted in Bcl2Δ51-85 contains several Ser and Thrresidues. It has been reported that Bcl-2 activity can be modulated byphosphorylation (34, 46, 47, and 49). Analysis of the activity ofseveral Bcl-2 mutants containing amino acid substitutions at Ser or Thrresidues, as described in Example 4, suggests that modulation of theproliferation-restraining activity by phosphorylation is possible.Alternative explanations to account for the mutant phenotype are alsopossible. The deleted region is rich in Ala and Pro residues.Substitution of Pro residues in two positions within the AP domainresulted in Bcl-2 mutants that permit enhanced cell proliferation. Thepossibility that these residues play some negative regulatory role inBcl-2 activity remains to be investigated.

In one aspect then, the invention provides isolated oligonucleotidesthat encode the Bcl-2 AP domain or fragments of the domain. Theoligonucleotides and short segments thereof are useful for screening formutations in the Bcl-2 AP domain by methods known in the art, such assingle strand conformational polymorphism (SSCP) and PCR mismatchanalysis.

In another aspect, the present invention is directed to identifyingprotein/protein interactions between the Bcl-2 AP domain and known or asyet unidentified cellular proteins. The Bcl-2 AP domain is also usefulin the identification and cloning of genes whose protein productsinteract with this domain in Bcl-2. The interacting proteins may play arole in modulation of cellular proliferation. The present invention alsorelates to an isolated polypeptide that is the Bcl-2 AP domain andfragments of the domain. The domain may be a target for allostericregulators of Bcl-2 function, such as protein kinases and/orphosphatases. Accordingly, peptides derived from this domain, preparedsynthetically or as bacterially expressed fusion proteins, can be usedas substrates to identify and characterize potential regulatory kinasesand/or phosphatases.

The invention further provides screening methods to identify moleculesthat modulate the proliferation-restraining activity of the AP domain.In one aspect, such screening methods involve the effect of a putativemodulating molecule on the short term or long term proliferation ofcells in culture expressing the AP domain. In another aspect, putativemodulating molecules can be identified by screening for agents thatdisrupt necessary protein/protein interactions mediated by the APdomain, using in vitro binding assays.

In yet another aspect, the invention provides for expression vectorscontaining genetic sequences, hosts transformed with such expressionvectors, and methods for producing the AP domain and fragments of thedomain that hinder or completely block proliferation.

In additional aspects, the present invention relates to antibodies thatspecifically bind to the AP domain and fragments of the domain thathinder or completely block cell proliferation. Peptides comprising thedomain are useful for producing antibodies thereto. Such antibodies areuseful for detecting and isolating proteins comprising the AP domain inbiological specimens including, for example, cells from all humantissues including heart tissue, lung tissue, tumor cells, brain tissue,placenta, liver, skeletal muscle, kidney, and pancreas, as well as formodulating the proliferation-restraining activity of proteins comprisingthe AP domain, in and from such biological specimens, and constituteadditional aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the domain structure of Bcl-2. The various conserveddomains (BH1-4) are indicated. BH1-3 are conserved among bothsurvival-promoting and death-promoting members of the Bcl-2 family ofproteins. BH1 and 2 are described in ref. 33. BH3 is described in ref.32. BH4 is conserved among survival-promoting members and corresponds tobox A described by Reed and coworkers (ref. 38). TM indicatestransmembrane domain. NH-1 indicates the E1B nineteen K homology domain(21). The amino acid sequence (SEQ ID NO:1) deleted in mutant Bcl2Δ51085is indicated.

FIGS. 2A to 2E illustrate suppression of p53-induced apoptosis by Bcl-2.FIG. 2A shows an immunoprecipitation analysis of Bcl-2 and Bcl2Δ51-85expression in BRK-p53val135-E1A cells. FIG. 2B is a graph showingsurvival/proliferation of BRK-p53val135-E1A cells at 32.5° C. , pRcCMVvector; ▪, wt Bcl-2; ▴, Bcl2Δ51-85. FIGS. 2C-E show the growth ofcolonies of cells transfected with vectors carrying various Bcl-2 genes.The Figures illustrate the long-term proliferation of BRK-p53val135-E1Acells. FIG. 2C, pRcCmV vector; FIG. 2D, wt Bcl2; FIG. 2E, Bcl2Δ51-85.

FIGS. 3A and 3B show suppression of Myc-induced apoptosis by Bcl-2. FIG.3A shows the results of an immunoprecipitation analysis of Bcl-2 andBcl2Δ51-85 expression in Rat1MycER-Hygro cells. FIG. 3B is a graphshowing survival/proliferation of RatMycER-Hygro cells. , pRcCMVvector; ▪, wt Bcl-2; ▴, Bcl2Δ51-85.

FIGS. 4A and B show the interaction of Bax with Bcl-2 and Bcl2Δ51-85.BSC40 cells were transfected with pTM-HA Bax and pTM-Bcl-2 orpTM-Bcl2Δ51-85 and infected with vaccinia virus vTF7-3. ³⁵ S-labeledproteins were immunoprecipitated either with HA mouse monoclonalantibody (FIG. 4A) or Bcl-2 rabbit polyclonal antibody (FIG. 4B) andanalyzed on 13% SDS-polyacrylamide gels.

FIG. 5 is a graph showing survival of BRK-p53val153-EIA cells expressingpoint mutants of Bcl-2. The number of viable cells was determined atvarious times after shifting to 32.5° C. by trypan blue exclusion and isplotted as a percentage of the number of live cells at the start of theexperiment.

DETAILED DESCRIPTION OF THE INVENTION

Isolated, in the context of the invention, indicates that someintervention occurs that increases the level of purity of a moleculeover that found in nature.

As mentioned above, the present invention is based upon the discovery ofa heretofore unidentified domain of the human Bcl-2 protein. This"anti-proliferation" or "AP" domain is required for modulation of cellproliferation. More specifically, the AP domain completely blocks cellproliferation.

The nucleotide sequence of human Bcl-2 according to this invention isbased on those described in reference 41, Genbank Accession #X06487 andin reference 42, Genbank Accession #M13994. The Bcl-2 nucleotidesequence described in reference 41 has a G at position 189 and an A atposition 287. The Bcl-2 nucleotide sequence described in reference 42contains an alternate nucleotide (C in place of G) at position 189 andan alternate nucleotide (G in place of A) at position 287 resulting inan amino acid change at residue 96 of Thr to Ala. The nucleotidesequence for Bcl-2 reported by Cleary et al (51), Genbank Accession#M14745, contains an alternate nucleotide at position 175 (A in place ofC), resulting in an amino acid change at residue 59 of Pro to Thr.

As used herein, the phrase "anti-proliferation (or AP) domain" means atruncated human Bcl-2 protein comprising amino acid residue 51 to any ofamino acid residues 85-97. (SEQ ID NOS:1-13).

Thus, in addition to the core residues, i. e. residues 51 to any ofamino acid residues 85-97, the AP domain can include stretches of 1 ormore amino acids in the amino-terminal direction from residue 85 and/or1 or more amino acids in the carboxyl-terminal direction from residue97, provided that the protein is truncated. That is, the AP domain ofthe present invention is not intended to include the full-length Bcl-2protein. For example, the AP domain can include the core residues, i. e., residue 51 to any of amino acid residues 85-97, and/or 5, 10, 15, 20residues, and so on, in increments of 5 to the amino-terminus of residue51 and/or carboxyl-terminus of residue 97.

The sequences of the polypeptide that make up the core residues, i. e.SEQ ID NOS:1-13, are set forth in the section following the examples.The sequences of any additional stretches upstream or downstream of thecore residues may be ascertained from the literature (E. G. 41, 42, 51)and protein databases such as EMBL.

Further, the data in Example 5 herein indicates that certain amino acidsare needed to maintain full or partial anti-proliferation activity ofthe AP domain. For example, any one of Ser at position 51, Pro atposition 57, Ser at position 62, Thr-Ser at positions 69 and 70, Thr atposition 74, and Pro at position 75 may contribute to theanti-proliferation function of the AP domain which is lost when residues51-85 are deleted. Thus, fragments of the AP domain that include any oneor more of the residues that fully or partially restoresanti-proliferation activity are within the present invention.

Functional equivalents of the polypeptide that make up the core residuesas defined by SEQ ID NOS: 1-13 are also within the present invention. By"functional equivalent" is meant a peptide possessing a biologicalactivity or immunological characteristic substantially similar to thatof the polypeptides that make up the core residues, and is intended toinclude "variants", "analogs", "homologs", or "chemical derivatives"possessing such activity or characteristics . Functional equivalents ofthe polypeptides that make up the core residues, then, may not share anidentical amino acid sequence, and conservative or non-conservativeamino acid substitutions of conventional or unconventional amino acidsare possible. However, in the present invention any of Ser at position51, Pro at position 57, Ser at position 62, Thr and Ser at positions 69and 70, Thr at position 74, or Pro at position 75, may not be Ala.

Reference herein to "conservative" amino acid substitution is intendedto mean the interchangeability of amino acid residues having similarside chains. For example, glycine, alanine, valine, leucine andisoleucine make up a group of amino acids having aliphatic side chains;serine and threonine are amino acids having aliphatic-hydroxyl sidechains; asparagine and glutamine are amino acids having amide-containingside chains; phenylalanine, tyrosine and tryptophan are amino acidshaving aromatic side chains; lysine, arginine and histidine are aminoacids having basic side chains; and cysteine and methionine are aminoacids having sulfur-containing side chains. Interchanging one amino acidfrom a given group with another amino acid from that same group would beconsidered a conservative substitution. Preferred conservativesubstitution groups include asparagine-glutamine, alanine-valine,lysine-arginine, phenylalanine-tyrosine and valine-leucine-isoleucine.

Functional equivalents that possess immunological characteristicssubstantially similar to that of the polypeptides that make up the coreresidues are useful, for example, as an antigen for raising antibodiesagainst the AP domain or fragments thereof or for detection orpurification of antibodies against the AP domain or fragments thereof.

The nucleotide sequences that encode the AP domain as defined herein arealso within the present invention. The nucleic acid compositions of theinvention will generally be in RNA or DNA forms, mixed polymeric forms,or any synthetic nucleotide structure capable of binding in abase-specific manner to a complementary strand of nucleic acid. Thedescribed nucleic acid embodiment is typically derived from genomic DNAor cDNA, prepared by synthesis, or derived from combinations thereof,including polymerase chain reaction (PCR) products.

The oligonucleotides that encode the core amino acids are those boundedby nucleotide 151 to any of nucleotides 255-291 (SEQ ID NO: 14-50),where nucleotide 1 is the first nucleotide of the codon encoding thefirst amino acid of Bcl-2. In these sequences the nucleotide at position175 can be C or A, the nucleotide at position 189 can be C or G, and thenucleotide at position 287 can be A or G.

The oligonucleotide sequences that encode the core residues, i. e. SEQID NOS: 14-50, are set forth in the section following the examples. ThecDNA sequences toward the 5' end of nucleotide 151 and toward the 3' endof nucleotide 291 may be ascertained from the literature (E. G. 22, 42,51) as well as from sequence databases such as Genbank.

Oligonucleotide fragments of oligonucleotide sequences that encode theAP domain are also included within the present invention and includefragments that contain at least one codon encoding an amino acid neededto maintain full or partial anti-proliferation activity of the APdomain. Examples are fragments that retain any one of the codons definedby nucleotides 151-153 (coding for Ser 51), 169-171 (coding for Pro 57),nucleotides 184-186 (coding for Ser 62), 204-210 (coding for Thr 69 andSer 70), 220-222 (coding for Thr 74), and 223-225 (coding for Pro 75).

The instant oligonucleotides and polypeptides may be obtained asdescribed herein, such as by recombinant means. For example, nucleotidesequences encoding the AP domain polypeptides or fragments thereof ofthe invention may be inserted into a suitable DNA vector, such as aplasmid, and the vector used to transform a suitable host. Therecombinant AP polypeptide or fragment is produced in the host byexpression. The transformed host may be a prokaryotic or eukaryoticcell, including a mammalian cell. The instant oligonucleotides andpolypeptides may also be used to obtain homologous nucleic acids andproteins by hybridization, for example, an instant nucleic acid can beused as a probe of a gene bank to identify clones with suitable homologytherewith. Also, within the confines of available technology, theoligonucleotides may be synthesized in vitro using, for example, solidphase oligonucleotide and oligopeptide synthetic methods known in theart.

The present invention also includes fusion polypeptides between the APdomain, or fragments thereof, or truncated wt Bcl-2 polypeptidesincluding the AP domain, and other proteins or polypeptides. Forexample, fusions may include proteins that serve as purificationtargets, such as, but not limited to glutathione S-transferase (GST)(43) and the FLAG epitope tag (Eastman Kodak). In addition, fusions mayinclude polypeptides that may have amino acid residues that have been orcan be chemically modified by phosphorylation, biotinylation, acylation,or other moieties, using methods known in the art. Fusion polypeptideswill typically be made by using either synthetic polypeptide orrecombinant nucleic acid methods known in the art.

The functional importance of the AP domain is related to its ability toregulate cell proliferation. This regulation may be mediated by one ormore protein/protein interactions between the domain and known Bcl-2interacting proteins such as Bax (44), Nip1-3 (29), Bik (32), Bak (31),R-ras (52), BAG-1 (45) and c-raf-1 (30) (see also Example 4) or as yetunidentified cellular proteins. The polypeptides of the presentinvention are useful to screen for proteins that interact with the APdomain, and these proteins and cDNA's encoding these proteins are alsopart of the invention. Such molecules are useful as agents formodulation of tumorigenesis and apoptotic activity of cells.

Methods for screening for proteins that interact with the AP domain arewell known in the art and include the yeast two-hybrid system (39, 28)and expression cloning strategies using recombinant fusion proteins.(53, 54)

The in vivo genetic strategy designated `two hybrid` cloning (39, 28)permits rapid genetic screening in yeast of molecules that associate,and the method has been used to isolate from expression libraries cDNAclones that code for proteins interacting with several known proteins.

Briefly, the method relies on the double transformation of yeast hostswith plasmids that encode fusion proteins. One plasmid carries partialsequences for a reporter molecule, for example, the GAL4 DNA bindingdomain, at the amino terminus of the fusion protein and sequences forthe known protein, to which a ligand is sought, also known as the "bait"at the carboxyl-terminus. For example, the bait can be the AP domainpolypeptide.

The second plasmid comprises sequences encoding a complementary proteinfor the reporter molecule, in the above case, required by the GAL4 DNAbinding domain, such as the GAL4 activation domain, at the aminoterminus and expressed products of individual cDNA from a bank at thecarboxyl-terminus. A suitable host is used to enable the selectionplanned. In the scenario discussed, the host would be one wherein theexpression of β-galactosidase is under the control of the GAL1 promoter.

Selection of double transformants are those that expressβ-galactosidase, hence would be blue colonies on an X-gal plate becausethe bait protein encoded by the cDNA of the second plasmid bind and thatinteraction juxtaposes the two GAL4 regulatory elements required forβ-galactosidase expression.

An additional related strategy is to isolate positive clones from thetwo hybrid assay that interact with GAL4 DNA-binding domain-Bcl-2 (wt)fusion but not with a GAL4 DNA-binding domain-Bcl-2Δ51-85 fusion. Suchinteracting proteins may require the identified domain for theirinteraction.

Thus, the present invention provides a method for screening for apolypeptide that binds the AP domain of Bcl-2 protein, the methodcomprising:

(a) conducting a double transformation wherein one vector expresses afusion protein comprising the AP domain or a fragment thereof and areporter molecule and the other vector expresses a fusion proteincomprising a complementary protein for the reporter molecule and thepolypeptide to be screened;

(b) monitoring for activation of the reporter molecule; and

(c) isolating cDNA that encodes the protein that binds to the AP domainor the fragment thereof,

wherein the AP domain or fragment thereof is a truncated Bcl-2 proteincomprising residues 51 to any of residues 85-97 (SEQ ID NOS:1-13) or afragment thereof that contains at least one amino acid needed tomaintain full or partial anti-proliferation activity of the AP domain.

In a related embodiment, the present invention also provides a method ofscreening for a polypeptide that binds the AP domain of Bcl-2 protein,the method comprising:

(a) conducting a first double transformation wherein one vectorexpresses a fusion protein comprising the AP domain and a reportermolecule and the other vector expresses a fusion protein comprising acomplementary protein for the reporter molecule and the polypeptide tobe screened;

(b) conducting a second double transformation wherein one vectorexpresses a fusion protein comprising Bcl-2 with the AP domain or afragment of Bcl-2 that contains at least one amino acid needed tomaintain full or partial anti-proliferation activity of the AP domaindeleted and a reporter molecule and the other vector expresses a fusionprotein comprising a complementary protein for the reporter molecule andthe polypeptide to be screened;

(c) monitoring for activation of the reporter molecule in both doubletransformations; and

(d) isolating cDNA that encodes a polypeptide that binds in step (a) butnot in step (b).

In a second example of methods of screening for proteins that interactwith the AP domain, a cDNA encoding Bcl-2 residues 51-85 is cloned intoan E. coli expression vector that will encode a glutathioneS-transferase (GST)-Bcl-2 domain fusion protein. The fusion protein isisolated following expression in bacteria and radiolabeled for use as aprobe to screen for cDNA of proteins capable of interacting with the APdomain from a human cell λ-phage expression library. (53, 54) Briefly, aλ-phage expression library (e.g.λ-ZAP, Stratagene) is plated on E. coliand resulting plaques are transferred to isopropyl-β-D-thogalactoside(IPTG)-impregnated nitrocellulose filters to induce protein expression.³² P-radiolabeled GST-AP domain fusion proteins or unlabelled GST-APdomain fusion proteins that can be detected with an anti-GST antibody,are used as a probe to screen for expressed proteins capable ofinteracting with the AP domain. Positive clones can be isolated and thegene encoding a protein capable of interacting with the AP domain can besequenced and characterized.

Thus, the present invention provides a method for screening for apolypeptide that interacts with the AP domain of Bcl-2 protein, themethod comprising:

(a) expressing cDNA that encodes a polypeptide to be screened;

(b) immobilizing the expressed polypeptide; and

(c) detecting interaction with a polypeptide comprising the AP domain orfragment thereof;

wherein the AP domain or fragment thereof is a truncated Bcl-2 proteincomprising residues 51 to any of residues 85-97 (SEQ ID NOS:1-13) or afragment thereof that contains at least one amino acid needed tomaintain full or partial anti-proliferation activity of the AP domain.

Alternatively, the biochemical isolation of interacting molecules isalso possible using isolated polypeptides comprising the Bcl-2 APdomain. For example, GST-AP domain fusion proteins can be immobilized onglutathione (GSH)-agarose columns to capture interacting proteins fromcell lysates. Cell lysates from BRK-p53val135-E1A cells are passed overthe column. Following washing to remove non-binding proteins,interacting proteins can be eluted using GSH or other conditions knownto disrupt protein/protein interactions such as salt, pH, guanidine HCl,or detergent gradients. Eluted proteins can be identified, for example,by SDS-PAGE and microsequencing. If necessary, oligonucleotide probesbased on the protein sequence can be used to clone the correspondinggene from an appropriate cDNA library.

Thus, the present invention provides a method for screening for apolypeptide that interacts with the AP domain of Bcl-2 protein, themethod comprising:

(a) immobilizing a polypeptide comprising the AP domain or fragment ofthe AP domain;

(b) contacting the immobilized polypeptide with putative interactingprotein; and

(c) identifying interacting protein;

wherein the AP domain or fragment thereof is a truncated Bcl-2 proteincomprising residues 51 to any of residues 85-97 (SEQ ID NOS:1-13) or afragment thereof that contains at least one amino acid needed tomaintain full or partial anti-proliferation activity of the AP domain.

The present invention includes the use of the AP domain or fragments forthe identification of agents that modulate AP domain mediated functions.Such agents may include peptides comprising the AP domain or mutants ofthe AP domain or comprising an AP domain. A "mutant" as used hereinrefers to a peptide having an amino acid sequence that differs from theamino acid sequence of the naturally occurring peptide or protein by atleast one amino acid. Mutants may have the same biological andimmununological activity as the naturally occurring AP domain. However,the biological or immunological activity of mutants may differ or belacking. Identification of such agents can be accomplished by thescreening of peptide or compound libraries, or other information banks,in assays for agonists or antagonists that enhance or inhibit AP domainfunction, e.g. survival-promoting and proliferation-restrainingactivity, as well as protein binding.

For example, BRK-p53val135-E1A cells expressing Bcl-2 or a truncatedversion of Bcl-2 comprising the AP domain can be used to screen foragents that inhibit the proliferation-restraining activity the AP domaindetected by increased proliferation in the short term assay and/orallowing colony formation in the long term assay.

In another example, agents can be identified that modulate theproliferation-restraining activity of the AP domain by screening forcompounds that influence protein/protein interactions mediated by the APdomain using an in vitro binding assay. In such as an assay, a GSTfusion protein comprising the AP domain is immobilized to GSH-agarose.Binding of a radiolabaled-interacting protein in the presence of one ormore compounds to be tested would be quantitated by scintillationcounting. Inhibitors of the interaction would result in a decrease inassociated interacting protein. For rapid-throughput screening, theGST/AP-domain fusion protein and biotinylated interacting protein areused in a multi-well plate format. Biotinylated proteins can beexpressed and isolated from E. coli using PinPoint vectors (Promega) byknown methods. The purified biotinylated protein is immobilized on aneutravidin-coated plate and binding of the GST/Ap-domain fusion proteinin the presence of test compounds is detected by ELISA using an anti-GSTmonoclonal antibody. Inhibitors of the interaction would score as adecreased ELISA signal.

A high speed screen using immobilized or "tagged" combinatoriallibraries can be used to identify agents that bind directly to the APdomain. Such agents are candidates to be tested for their ability toenhance or inhibit the proliferation-restraining activity of Bcl-2.

The AP domain may be a target for allosteric regulators of Bcl-2function such as protein kinases and/or phosphatases. Phosphorylation ofBcl-2 has been reported (46-49) and it has been suggested thatphosphorylation/dephosphorylation may play a role in the regulation ofBcl-2 function. The identified domain of Bcl-2 contains severalpotential phosphorylation sites. Thus, the polypeptides of the presentinvention comprising the AP domain can be used as substrates to measurean enzymatic activity, such as kinase or phosphatase. In this aspect, invitro kinase assays are carried out by incubating cell lysates, such asderived from BRK-p53val135-E1A cells, with AP domain polypeptides,prepared synthetically or as bacterially expressed fusion proteins, inthe presence of ²³ P-labeled ATP in 10 mM Tris buffer containing 10 mMMgCl₂ and 1 μM unlabeled ATP. Phosphatase activity is detected byincubating cell lysates with phosphorylated AP domain polypeptide,derived from in vitro kinase assays described above or isolated fromcells, and following the release of radiolabeled phosphate from the APdomain. Purification and sequencing of the protein responsible for thisactivity can be accomplished by standard methods such as those describedin "Protein Purification: Principles and Practice," by Robert Scopes(Ed: C. Cantor, Springer Verlag, Heidelberg, 1982).

Synthetic peptides or fusion proteins containing this domain can be usedfor immunizing animals in the production of polyclonal or monoclonalantibodies that bind to this domain in Bcl-2. Such antibodies would beuseful as reagents for studying the function of this domain. Forexample, microinjection of anti-domain antibodies may alter the cellcycle arrest activity of Bcl-2. Such antibodies may also prove to beuseful in screening for mutations in this domain of Bcl-2 that causealterations in antibody binding. These mutations may correlate withalterations in Bcl-2 function.

The AP polypeptides of the invention also may be used for the detectionof Bcl-2 by means of standard assays including radioimmunoassays andenzyme immunoassays.

The polypeptides of the present invention or fusion proteins thereof arealso useful to make antibodies for detection or determination ofproteins comprising the AP domain, for example, in fractions fromtissue/organ excisions, by means of immunochemical or other techniquesin view of the antigenic properties thereof. Immunization of animalswith polypeptides comprising the AP domain alone or in conjunction withadjuvants by known methods can produce antibodies specific for the APdomain polypeptide. Antiserum obtained by conventional procedures may beutilized for this purpose. For example, a mammal, such as a rabbit, maybe immunized with a peptide comprising the AP domain, thereby inducingthe formation of polyclonal antibodies thereagainst. Monoclonalantibodies also may be generated using known procedures.

If the target molecule is poorly immunogenic, known methods forenhancing immunogenicity, such as, use of adjuvants, use of fragments ofthe target molecule as antigen, conjugating the target molecule orfragments thereof to a known carrier, such as albumin or keyhole limpethemocyanin, immunizing immune cells in vitro and the like, as known inthe art can be used.

Antibodies against the AP domain polypeptides or fragments thereof ofthe invention may be used to screen cDNA expression libraries foridentifying clones containing cDNA inserts encoding structurallyrelated, immunocrossreactive proteins that may be members of an APdomain family of proteins. Screening of cDNA and mRNA expressionlibraries is known in the art. Similarly, antibodies against AP domainpolypeptides or fragments thereof can be used to identify or purifyimmunocrossreactive proteins related to this domain, or to detect ordetermine the amount of proteins containing the AP domain in a cell orcell population, for example, in tissue or cells, such as lymphocytes,obtained from a patient. Known methods for such measurements includeimmunopreciptiation of cell extracts followed by PAGE, in situ detectionby immunohistochemical methods, and ELISA methods, all of which are wellknow in the art. In addition, antibodies against the AP domain orfragments thereof may be used to modulate the proliferation-restrainingactivity of proteins comprising the AP domain.

Accordingly, the present invention also provides an isolated antibodythat binds to the AP domain of Bcl-2 and a hybridoma that makesmonoclonal antibody that specifically binds to the AP domain.

The cDNA of the present invention may be used for screening formutations in the AP domain in, for example, human tumors. Indeed,mutations within this domain associated with non-Hodgkin's lymphomashave been reported including a change in the nucleotide (T in place ofC) at position 175 resulting in a substitution of Pro 59 with Ser (50).

Methods for screening for such mutations have been described, andinclude single strand conformational polymorphism (SSCP) of polymerasechain reaction-amplified DNA fragments (SSPC-PCR) (55, 56) andPCR-mismatch analysis (50, 51).

In SSCP-PCR, oligonucleotide primers are used to amplify the segment ofthe Bcl-2 gene encoding the AP domain from DNA or mRNA isolated from atest sample or from cDNA made from the test sample. The PCR product isthen heat denatured, subjected to electrophoresis on polyacrylamide gelsand transferred to a nylon membrane. The fragment can be detected by achemiluminescence detection system and the relative mobility of the testfragment with a control fragment from wt Bcl-2 is determined. A singlebase change can be detected by this method.

Accordingly, the present invention provides a method of screening formutations in the AP domain of Bcl-2, the method comprising:

(a) isolating genomic DNA, cDNA or mRNA from a specimen to be screened;

(b) amplifying DNA fragments encoding the AP domain or portions thereoffrom the genomic DNA, cDNA or mRNA;

(c) denaturing the amplified product;

(d) subjecting the denatured product to electrophoresis; and

(e) detecting mutations by comparing the mobility of the denaturedamplified product to a control DNA encoding the AP domain or portionsthereof corresponding positionally to the DNA fragments amplified instep (b);

wherein the control DNA encoding the AP domain or portions thereof isfrom the truncated cDNA encoding the bcl-2 gene and fragments of thetruncated cDNA.

Alternatively, in PCR-mismatch analysis, PCR products from the testsample are mixed with radiolabeled PCR products from the wild type Bcl-2AP domain . The mixed PCR material is denatured and then annealed.Chemical modification and cleavage of heteroduplexes containingmismatched nucleotides is analyzed by gel electrophoresis. PCR-generatedDNAs containing mutations are then subcloned and sequenced to identifythe precise nature of the mutation.

Thus, the present invention provides a method of screening for mutationsin the AP domain of Bcl-2, said method comprising:

(a) isolating genomic DNA, cDNA or mRNA from a specimen to be screened;

(b) amplifying DNA fragments encoding the AP domain or portions thereoffrom the genomic DNA, cDNA or mRNA;

(c) mixing the amplified product with labeled PCR product from thecorresponding position in a control AP domain or portion thereof;

(d) denaturing and annealing the mixed PCR products; and

(e) analyzing for mismatched nucleotides by electrophoresis followingchemical modification;

wherein the control DNA encoding the AP domain or portions thereof isselected from the truncated cDNA encoding the bcl-2 gene and fragmentsof the truncated cDNA.

In a related embodiment, the present invention also provides a method ofscreening for mutations in the AP domain of Bcl-2, said methodcomprising:

(a) isolating genomic DNA, cDNA or mRNA from a specimen to be screened;

(b) amplifying DNA fragments encoding the AP domain or portions thereoffrom the genomic DNA, cDNA or mRNA; and

(c) sequencing the amplified DNA product.

Of course, the polynucleotide sequences of the invention may be used inthe PCR method to detect the presence of mRNA encoding AP domainpolypeptides in for, example, cells from all human tissues includingheart tissue, lung tissue, tumor cells, brain tissue, placenta, liver,skeletal muscle, kidney, and pancreas.

EXAMPLES

The invention will now be described by means of working examples thatare not intended to be limiting.

Materials and Methods

Plasmids. Plasmid pRcCMV-Bcl-2 was constructed by cloning the humanbcl-2 gene (22) into the HindIII and Xbal sites of the mammalianexpression vector pRcCMV (Invitrogen). Mutant Bcl2Δ51-85 was constructedby PCR mutagenesis using a mutagenic oligonucleotide primer5'-GGA-CCA-CAG-GTG-GCA-CCG-GGC-TGA-GGC-TAG-CGG-AGA-AGA-AGC-CCG-GTG-CGG-GGG-CG-3'(SEQ ID NO:51) and two other primers complementary to the 5' and 3' endsof bcl-2. This mutagenesis introduces an Nhel site, and substitutes analanine and a serine residue in the deleted region. The PCR product wascloned into the HindIII and Xbal sites of pRcCMV to generatepRcCMV-Bcl2Δ51-85. pTM1-based plasmids expressing wt Bcl-2 and mutantBcl2Δ51-85 were constructed by cloning the respective genes into theNcol and Sall sites of the vector pTM1 (23).

Cell lines. The BRK-p53val135-E1A cell line has been described (21) andwas maintained at 38.5° C. in Dulbecco's modified Eagle medium (DMEM)supplemented with 10% fetal calf serum. BRK-p53val135-E1A cells stablyexpressing Bcl-2 were generated by transfection of various pRcCMV-basedBcl-2 expression plasmids and selection with G418 (250μg/ml)(GIBCO/BRL). Rat1a and Rat1MycER-Hygro cells have been described(14,24). Cells expressing ER fusion proteins were maintained in DMEMmedia without phenol red and 10% fetal calf serum (certified lowestrogen content, GIBCO/BRL). Rat1MycER-Hygro cells expressing Bcl-2were selected by transfection with pRcCMV-Bcl2 or pRcCMV-Bcl2Δ51-85 andselection with 400 μg/ml G418. DNA transfections were carried out by thestandard calcium phosphate method.

Cell death assays. BRK-p53val135-E1A cells were plated at 5×10⁵ cells/35mm dish. After 12 hours at 38.5° C., the dishes were shifted to 32.5°C., and at various intervals cells were trypsinized in triplicates,stained with 0.2% trypan blue and viable cells were counted. Similarly5×10⁵ Rat1-Hygro cells were plated in 35 mm dishes, incubated for 12hours at 37° C., washed three times in serum-free DMEM and maintained infresh media containing 0.1% fetal calf serum and 1 μM β-estradiol.Viable cell number was determined at various intervals.

Immunoprecipitation. Bcl2 or Bcl2Δ51-85 proteins were co-expressed withHA epitope-tagged Bax using the vaccinia virus/T7 coupled expressionsystem as previously described (29). BSC40 cells were transfected withpTM1 expression plasmids using LipofectAMINE (GIBCO/BRL) and infectedwith the recombinant vaccinia virus vTF7-3 (23) expressing the T7 RNApolymerase. Sixteen hours post-infection, cells were metabolicallylabeled with 500 μCi of ³⁵ S-methionine and -cysteine mixture for twohours and lysed in isotonic buffer (29) containing protease inhibitors(0.04 mg/ml aprotinin, 0.2 mg/ml leupeptin, 200 μM phenylmethylsulfonylfluoride). Lysates were precleared with protein A-Sepharose for 1 hour,which was removed by centrifugation. The proteins wereimmunoprecipitated with a rabbit polyclonal antibody specific for humanBcl-2 or with HA monoclonal antibody (12CA5; Boehringer Mannheim). Theproteins were analyzed by electrophoresis on 13% SDS polyacrylamide gelsand detected by fluorography.

Example 1 Effect on p53-Induced Apoptosis

A non-conserved region located between residues 51 and 85 was examined(FIG. 1) with the rationale that such sequences may regulate theactivity of Bcl-2. Deletion of this region of Bcl-2 (Bcl 2Δ51-85) didnot significantly alter the level of expression of the mutant protein(FIG. 2A; 3A). The effect of Bcl-2 wt and mutant Bcl2Δ51-85 on apoptosisinduced by the tumor suppressor protein p53 (25) was tested. Baby ratkidney (BRK) cells transformed with adenovirus E1A and a ts mutant ofp53 (p53val135) (26) express very high levels of mutant p53 at thenon-permissive (38.5 C.) temperature and undergo rapid apoptosis afterthe p53 protein assumes wt conformation at 32.5° C. (27). This apoptosiscan be efficiently suppressed by Bcl-2 (20). BRK-p53val135-E1A cellswere transfected with pRcCMV vector or pRcCMV-Bcl-2 or pRcCMV-Bcl2Δ51-85and G418 resistant colonies were selected at 38.5° C. As expected, wtBcl-2 efficiently suppressed cell death compared to cells transfectedwith pRcCMV vector (FIG. 2A). Cells expressing Bcl2Δ51-85 did not losecell viability significantly at 32.5° C. Surprisingly, however, thesecells also proliferated efficiently at this temperature in contrast tocells expressing wt Bcl-2 (FIG. 2B). Deletion of additional residues,form 51-98, resulted in a mutant, Bcl-2Δ51-98 that was unable tosuppress cell death in this assay (FIG. 5, Table 1) suggesting thatresidues between 85 and 98 may be critical for Bcl-2 survival function.

The effect of mutant Bcl2Δ51-85 on long term proliferation was alsodetermined. Pooled cell lines transfected with Bcl-2 wt or Bcl2Δ51-85 orpRcCMV vector were plated at low cell density, maintained at 32.5° C.for three weeks and stained with Giemsa (FIGS. 2C-2E). Cells tranfectedwith pRcCMV died rapidly without forming any detectable colonies. Cellstranfected with wt Bcl-2 survived for an extended period, but formedvery few proliferating colonies. Consistent with their behavior in shortterm cell survival/proliferation assays (FIG. 2B), cells transfectedwith mutant Bcl2Δ51-85 formed numerous proliferating colonies. Theseresults indicate that the mutant Bcl2Δ51-85 facilitates long termproliferation of cells under conditions that otherwise result inapoptosis.

Example 2 Effect on Myc-Induced Apoptosis

The effect of Bcl2Δ51-85 on Myc-induced apoptosis was also tested. Rat1cells expressing the c-myc gene fused to the human estrogen receptor(c-mycER-Hygro) undergo apoptosis after Myc expression is activated byaddition of β-estradiol and cells are deprived of serum (13,14). Thec-mycER-Hygro cells were tranfected with pRcCMV vector or pRcCMV-basedplasmids expressing wt Bcl-2 or mutant Bcl2Δ51-85 and pooledG418-resistant cell lines were established. Immunoprecipitation (FIG.3A) and protein-blot (not shown) analyses revealed that the various Rat1cell lines expressed comparable levels of wt or the mutant Bcl-2proteins. The effect of Bcl-2 expression on Myc-induced apoptosis wasthen determined by treating the cells with 1 μM β-estradiol in mediacontaining 0.1% fetal calf serum. Deregulated Myc expression inducedsignificant cell death. Expression of wt Bcl-2 resulted in about 60%cell survival. As in the case of BRK/p53val135-E1A cells, the expressionof Bcl2Δ51-85 mutant not only suppressed cell death but also inducedsignificant proliferation on mycER-Hygro cells in low serum after a lagperiod of about one day.

Example 3 Interaction of Cellular Proteins with Bcl2Δ51-85

In order to determine if deletion of the amino acid region encompassingresidues 51-85 affected interaction of various cellular proteins, theinteraction of several cellular proteins that have been previouslyreported to interact with Bcl-2 either by two-hybrid interaction studiesin yeast (28) or by co-immunoprecipitation analyses was examined. Inthese studies, no major difference was observed in the patterns ofinteraction of Nip1-3 (29), c-Raf-1 (30), R-ras (52), Bak (31), and Bik(32) (not shown). In contrast, the level of interaction between Bax (33)and Bcl2Δ51-85 appeared to be significantly enhanced in comparison to wtBcl-2 in co-immunoprecipitation assays (FIGS. 4A and 4B). This enhancedinteraction appears to be significant considering that the total levelof Bax was similar in cells expressing either Bcl2Δ51-85 or wt Bcl-2.

Example 4 Characterization of Critical Residues within The Bcl-2 Residue51-85 Domain

In an effort to characterize critical residues within the Bcl-2 residue51-85 domain, several Bcl-2 mutants encoding single amino acidsubstitutions were constructed and tested for their effect on cellsurvival and proliferation. In the short term survival assay (FIG. 5 andTable 1), none of the point mutants gave an enhanced proliferationactivity comparable to the Bcl-2D51-85 mutant, though two mutants, P75Aand S51A, had some effect. While most of the point mutations resulted inBcl-2 molecules that retained at least significant survival function,substitution of serine at position 62 with alanine completely abolishedsurvival activity. This result demonstrates that this region hassubstantial influence on the survival function of Bcl-2 as well asmodulation of proliferation. In the long term assay (Table 1), severalof the point mutants permitted significant colony formation, suggestingthat these residues may contribute to the proliferation-restrainingactivity of Bcl-2. One substitution mutant, S51A, had a hyperprotectiveeffect that was apparent in the long term assay. With wild type Bcl-2,the BRK-p53val135-E1A cells eventually die when subjected to theprolonged exposure at 32.5° C. used in the long term assay. In contrast,cells expressing Bcl-2 S51A survived for the duration of the assay,though no significant colony formation was observed.

                  TABLE 1                                                         ______________________________________                                        Comparison of survival activity and long term colony formation for            Bcl-2 mutants. Survival activity and long-term proliferation (colony          formation) was measured in BRK-p53val135-E1a cells at 32.5° C. as      described for FIG. 2. Δ indicates deleted residues, substitution        mutations (such as S51A) are indicated by the amino acid changed              followed by the position number and the substituted amino acid. For           survival activity, + is normal +\- is partial, ++ and               + + + are above normal. For colony formation, + is small, + + is              medium, +++ is large colonies. Mat indicates that cells were present          without obvious colony formation. -, indicates no cells remaining.                            Survival                                                                              colony                                                Bcl-2 mutant    Activity                                                                              formation                                             ______________________________________                                        vector          -       -                                                     wild type       +       -                                                     Δ51-85    ++      +++                                                   Δ51-98    -       -                                                     S51A            +++     mat                                                   T56A            +\-                                                                         -                                                     P57A            +\-                                                                         +                                                     S62A            -       -                                                     TS69-70AA       +\-                                                                         +                                                     T74A            +\-                                                                         ++                                                    P75A            ++      +                                                     ______________________________________                                    

A straightforward interpretation of these results is that the effect ofthe Bcl-2Δ51-85 mutant is a sum of the hyperproliferative function ofthe S51A mutant and the proliferative effects of the P57A, TS69-70AA,T74A, and P75A mutants.

SEQUENCES

Polypeptide and nucleotide sequences referred to herein by SEQ ID NOS.are listed below.

In the polypeptide sequences (Pro/Thr) and (Thr/Ala) means that theamino acid at that position can be Pro or Thr and Thr or Ala,respectively.

In the nucleic acid sequences M represents A or C, S represents C or G,and R represents A or G. ##STR1##

REFERENCES

1. Y. Tsujimoto et al., Science, 228: 1440-1443, 1985.

2. A. Bakhshi et al., Cell, 41: 899-906, 1985.

3. M. L. Cleary et al., Cell, 47: 19-28, 1986.

4. T. J. McDonnell et al., Cancer Res., 52: 6940-6944, 1992.

5. F. Pezzella et al., N. Engl. J. Med., 329: 690-694, 1994.

6. Q. L. Lu et al., Internatl. J. Cancer, 53: 29-35, 1993.

7. S. J. Korsmeyer, Blood, 80: 879-886, 1992.

8. J. C. Reed, J. Cell. Biol., 124: 1-6, 1994.

9. C. J. Hawkins and D. L. Vaux, Immunol. Reviews, 142: 127-139, 1994.

10. G. T. Williams, Cell, 65: 1097-1098, 1991.

11. D. L. Vaux et al., Nature, 335: 440-442, 1988.

12. D. S. Askew et al., Oncogene, 6: 1915-1922, 1991.

13. G. I. Evan et al., Cell, 69: 119-128, 1992.

14. A. J. Wagner et al., Mol. Cell. Biol., 13: 2432-2440, 1993.

15. R. P. Bissonnette et al., Nature, 359: 552-554, 1992.

16. A. Fanidi et al., Nature, 359, 554-556, 1992.

17. J. J. Ryan et al., Proc. Natl. Acad. Sci. U.S.A., 91: 5878-5882,1994.

18. T. Miyashita et al., Cancer Res., 54: 3131-3135, 1994.

19. M. Selvakumaran et al., Oncogene, 9: 1791-1798, 1994.

20. S. K. Chiou, et al., Mol. Cell. Biol., 14: 2556-2563, 1994.

21. T. Subramanian, et al., Oncogene, 11: 2404-2409, 1995.

22. M. Seto et al., EMBO, 7: 123-131, 1988.

23. T. R. Fuerst et al., Mol. Cell. Biol., 7: 2538-2544, 1987.

24. A. J. Wagner et al., Genes Dev., 8: 2817-2830, 1994.

25. M. Oren, Seminars Cancer Biol., 5: 221-227, 1994.

26. D. Michalovitz et al., Cell, 62: 671-680, 1990.

27. M. Debbas and E. White, Genes Dev., 7: 546-554, 1993.

28. C. T. Chien et al., Proc. Natl. Acad. Sci. USA, 88: 9578-9582, 1991.

29. J. M. Boyd et al., Cell, 79: 341-351, 1994.

30. H G. Wang et al., Oncogene, 9: 2751-2756, 1994.

31. T. Chittenden et al., Nature, 374: 733-736, 1995.

32. J. M. Boyd et al., Oncogene, 11: 1921-1928, 1995.

33. Z. N. Oltvai et al., Cell, 74: 609-619, 1993.

34. S. Haldar et al., Proc. Natl. Acad. Sci. USA, 92: 4507-4511, 1995.

35. S. Henderson et al., Proc. Natl . Acad. Sci. USA, 90: 8479-8483,1993.

36. B. Tarodi et al., Virology, 201: 404-407, 1994.

37. S. Takayama et al., DNA and Cell Biol., 13: 679-692, 1994.

38. T. Sato et al., Proc. Natl. Acad. Sci. USA, 91: 9238-9242, 1994.

39. S. Fields and O. K. Song, Nature, 340: 245-246, 1989.

40. J. A. Pietenpol, Cancer Research, 54: 3714-3717, 1994.

41. M. Seto et al., The EMBO Journal, 7: 123-131, 1988.

42. Y. Tsujimoto et al., Proc. Natl. Acad. Sci. U.S.A., 83: 5214-5218,1986.

43. D. B. Smith et al., Gene, 67: 31-40, 1988.

44. Z. N. Oltvai et al., Cell, 74: 609-619, 1993.

45. S. Takayama et al., Cell, 80: 279-284, 1995.

46. C. -Y Chen and D. V. Faller, Oncogene, 11: 1487-1498, 1995.

47. C. -Y Chen and D. V. Faller, J. Biol. Chem., 271: 2376-2379, 1996.

48. S. Haldar et al., Proc. Natl. Acad. Sci. U.S.A., 92: 4507-4511,1995.

49. W. S. May et al., J. Biol. Chem., 269: 26865-26870, 1994.

50. S. Tanaka et al., Blood, 79: 229-237, 1992.

51. M. L. Cleary et al., Cell, 47: 19-28, 1986.

52. M. J. Fernanadez-Sarabia and J. R. Bischoff, Nature, 366: 274-275,1993.

53. E. Y. Skolnik et al., Cell, 65: 83-90, 1991.

54. W. G. Kaelin et al., Cell, 70: 351-364, 1992.

55. M. Orita et al., Genomics, 5: 874-879, 1989.

56. M. Orita et al., Proc. Natl. Acad. Sci. U.S.A., 86: 2766-2770, 1989.

57. A. J. Montandon et al., Nucleic Acids Res., 17: 3347, 1989.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 51                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAla                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAlaLeu                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAlaLeuSer                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAlaLeuSerPro                                                            35                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAlaLeuSerProVal                                                         35                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAlaLeuSerProValPro                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAlaLeuSerProValProPro                                                   3540                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAlaLeuSerProValProProVal                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAlaLeuSerProValProProValVal                                             3540                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAlaLeuSerProValProProValValHis                                          3540                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAlaLeuSerProValProProValValHisLeu                                       354045                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAlaLeuSerProValProProValValHisLeuXaa                                    354045                                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      SerGlnProGlyHisThrProHisXaaAlaAlaSerArgAspProVal                              151015                                                                        AlaArgThrSerProLeuGlnThrProAlaAlaProGlyAlaAlaAla                              202530                                                                        GlyProAlaLeuSerProValProProValValHisLeuXaaLeu                                 354045                                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 104 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGC104                               (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 105 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCG105                              (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 106 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGC106                             (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCT107                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTC108                           (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 109 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCA109                          (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 110 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAG110                         (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 111 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGC111                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 112 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCC112                       (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 113 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCC113                      (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 114 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCG114                     (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 115 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGG115                    (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGT116                   (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 117 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTG117                  (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 118 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGC118                 (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 119 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCC119                (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 121 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               C121                                                                          (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 122 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CC122                                                                         (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 123 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCT123                                                                        (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 124 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTG124                                                                       (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 125 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGT125                                                                      (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 126 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTG126                                                                     (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 128 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGT128                                                                   (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 129 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTC129                                                                  (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 130 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTCC130                                                                 (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 131 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTCCA131                                                                (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 132 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTCCAC132                                                               (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 133 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTCCACC133                                                              (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 134 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTCCACCT134                                                             (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 135 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTCCACCTG135                                                            (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 136 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTCCACCTGR136                                                           (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 137 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTCCACCTGRC137                                                          (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 138 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTCCACCTGRCC138                                                         (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 139 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTCCACCTGRCCC139                                                        (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 140 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTCCACCTGRCCCT140                                                       (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 141 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      TCCCAGCCCGGGCACACGCCCCATMCAGCCGCATCCCGSGACCCGGTCGCCAGGACCTCG60                CCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCA120               CCTGTGGTCCACCTGRCCCTC141                                                      (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Oligonucleotide Primer"                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      GGACCACAGGTGGCACCGGGCTGAGGCTAGCGGAGAAGAAGCCCGGTGCGGGGGCG56                    __________________________________________________________________________

What is claimed:
 1. An isolated bcl-2 polynucleotide consisting ofnucleotides 151 to any one of nucleotides 255-291 (SEQ ID NOS:14-50), orfragments of said isolated bcl-2 polynucleotide that contain at leastone of nucleotides 151-153, nucleotides 169-171, nucleotides 184-186,nucleotides 205-210, nucleotides 220-222, and nucleotides 223-225,wherein said fragments encode peptides which exhibit full or partialanti-proliferation activity of the AP domain.
 2. The isolated bcl-2polynucleotide of claim 1, which consists of nucleotides 151 to any oneof nucleotides 255-291 (SEQ ID NOS:14-50).
 3. The isolated bcl-2polynucleotide of claim 1, which consists of nucleotides 151-255 (SEQNO:14).
 4. RNA complementary to said isolated bcl-2 polynucleotide orfragments of any one of claims 1, 2, or
 3. 5. An isolated Bcl-2polypeptide consisting of residues 51 to any of 85-97 (SEQ ID NOS:1-13)or fragments of said isolated Bcl-2 polypeptide that contain at leastone of Ser residue 51, Pro residue 57, Ser residue 62, Thr and Serresidues 69 and 70, Thr residue 74, and Pro residue 75, wherein saidfragments exhibit full or partial anti-proliferation activity of the APdomain.
 6. An isolated Bcl-2 polypeptide of claim 5 consisting ofresidues 51-85 (SEQ ID NO:1) or fragments of said isolated Bcl-2polypeptide consisting of residues 51-85 that contain at least one ofSer residue 51, Pro residue 57, Ser residue 62, Thr and Ser residues 69and 70, Thr residue 74, and Pro residue
 75. 7. An isolated Bcl-2polypeptide of claim 5 which consists of residues 51 to any of 85-97(SEQ ID NOS:1-13).
 8. An isolated Bcl-2 polypeptide of claim 5 whichconsists of residues 51-85 (SEQ ID NO:1).
 9. A method of producingisolated Bcl-2 polypeptides consisting of residues 51 to any of 85-97(SEQ ID NOS:1-13) or fragments of said isolated Bcl-2 polypeptides thatcontain at least one of Ser residue 51, Pro residue 57, Ser residue 62,Thr and Ser residues 69 and 70, Thr residue 74, and Pro residue 75,wherein said fragments exhibit full or partial anti-proliferationactivity of the AP domain, said method comprising:(a) constructing avector comprising DNA encoding said Bcl-2 polypeptides or said fragmentsthereof that exhibit full or partial anti-proliferation activity of theAP domain; (b) transforming a suitable host cell with said vector ofstep (a); (c) culturing said host cell under conditions that allowexpression of said Bcl-2 polypeptides or said fragments thereof by saidhost cell; and (d) isolating said Bcl-2 polypeptides or said fragmentsthereof expressed by said host cell of step (c).
 10. The method of claim9, wherein said host cell is a mammalian cell.